Pseudomonas aeruginosa is an important opportunistic pathogen both in terms of the morbidity and mortality of infections it causes. A significant proportion of patients with cystic fibrosis (CF), are colonized at an early age (l-2yrs) with this organism and most CF patients ultimately succumb to a chronic lung infection from P. aeruginosa. The reason for the extraordinary pathogenicity of P. aeruginosa in these patients, as compared to other Pseudomonads for example, is not clear. It is highly probable that the myriad of virulence determinants that P. aeruginosa produces contributes to its pathogenic potential. Unfortunately, the exact contribution of these factors, alone or in combination, to even the simplest kind of P. aeruginosa infection has not yet been elucidated. Moreover, the fact that expression of essentially all of the identified major virulence determinants is regulated by a variety of environmental conditions which the organism may encounter in the host adds a great deal of complexity to analyzing the pathogenicity of P. aeruginosa. In the past few years studies using molecular, biochemical and genetic approaches have begun to elucidate the mechanisms of regulation of virulence determinants. A more comprehensive understanding of regulatory processes involved in the expression of virulence determinants could help in the rational development of effective measures for the prevention and therapy of all kinds of P. aeruginosa infections. The research described in this proposal is designed to fill gaps in our knowledge regarding two specific issues salient to the pathogenic potential of P. aeruginosa. The first relates to the mechanisms of how environmental signals, specifically the concentration of environmental iron and oxygen, regulate the expression of virulence determinants of P. aeruginosa, particularly exotoxin A and protease. The mechanism by which iron regulates the expression of these gene products is not completely understood. Furthermore, while it has been known for many years that oxygen regulates the expression of ETA, essentially nothing is known about this mechanism of regulation. This oxygen regulation is particularly relevant to the pathogenesis of P. aeruginosa in lung infections (e.g. in CF patients) where oxygen therapy could regrettably enhance the expression of virulence determinants such as ETA. The second issue to be addressed by this research pertains to the isolation and further characterization of insertion sequence (IS) elements that are unique to P. aeruginosa and are localized to a region of the genome adjacent to the 5' end of the exotoxin A gene (toxA). There is only a limited amount of information available regarding the biology of IS elements unique P. aeruginosa, and essentially nothing is known regarding their potential role in the expression of pathogenicity. Molecular, genetic and biochemical approaches described in this proposal will be used to examine both questions that are significant to the pathogenesis of P. aeruginosa.